1. Field of the Invention
The present invention relates to a method and assay kit for the analysis of the oxidative modification of protein-containing substances and of the oxidative stress in biological samples by measuring the antiradical properties of their protein-containing components.
2. Discussion of the Background
Oxidative stress is a common phenomenon which is implicated in the etiopathogenesis of several diseases such as atherosclerosis, cancer, acute inflammation, etc. Methods determining the concentration of species reactive with thiobarbituric acid (TBA) or of conjugated dienes have been used as routine measurements for the determination of the degree of severity of oxidative stress. Products of lipid peroxidation, for example, malondialdehyde and 4-hydroxynonenal are reactive with thiobarbituric acid.
One drawback of the known methods for the determination of oxidative stress is the lack of specificity, because several substances react with thiobarbituric acid. Another drawback is the relative insensitivity because lipid peroxidation does not immediately accompany oxidative stress. In fact, lipid peroxidation occurs only after antioxidants have been exhausted (see FAVIER, A.: Oxidative stress: value of its demonstration in medical biology and problems posed by the choice of a marker, Ann. Biol. Clin. (Paris), Vol. 55, 1997, pp. 9-16).
Proteins, unlike lipids, react immediately to oxidative stress. Different alterations, particularly in amino acids are detected in protein degradation assays (see PACIFICI, Robert E.; DAVIES, Kelvin J. A.: Protein Degradation as an Index of Oxidative Stress. In: METHODS IN ENZYMOLOGY, Vol. 186, Part B, Eds. Packer, L. and Glazer, A. N. Academic Press, Inc. 1990, pp. 485-502). These alterations include formation of characteristic products, alterations in the secondary, tertiary and quaternary structure, electric charge, folding, hydrophobicity, fragmentation, covalent inter- and intramolecular cross-linkage or increase in proteolytic sensitivity. However, determination of these parameters is very complicated, expensive, cumbersome and often non-specific, requiring methods such as radioactive or fluorescent labeling, gel electrophoresis, Western blots and immunoprecipitation.
Accordingly, there has been a need for a simplified method that allows determination of oxidative stress in organisms and the evaluation of antiradical activity of substances, particularly without the interference of low-molecular weight antioxidants, such as ascorbic acid and uric acid.
It is an objective of the present invention to devise a new method for the determination of oxidative stress in an organism. Another objective is to devise an assay kit for the measurement of oxidative stress in an organism by investigation of body fluids, for example blood plasma.
These and other objects are achieved according to the invention, the first embodiment of which includes a method for quantitative analysis of the oxidative modification of a protein-containing substance, comprising:
purifying said protein-containing substance, thereby removing a low-molecular weight antioxidant and providing a purified protein-containing substance;
generating free radicals in said purified protein-containing substance;
measuring the antiradical properties of said purified protein-containing substance in a free-radical generating measuring system.
Another embodiment of the invention includes an assay kit for the analysis of oxidative modification of protein-containing substances, comprising:
a gel chromatographic column, an aqueous photosensitizer solution and an aqueous carbonate buffer solution.
FIG. 1:
Antioxidant capacity (ACW) of methionine, measured in a 2 mmol/l solution in H2O (lower curve) and human serum albumin (HSA) in a 60 g/l solution in H2O (upper curve) during irradiation with UV light (xcex=254 nm) in equivalent concentration of ascorbic acid (calibration substance).
FIG. 2:
Antioxidant capacity of histidine (2 mmol/l) under conditions of chemical (NaOCl) and physical (UV, xcex=254 nm) oxidation in equivalent concentration of ascorbic acid (calibration substance).
For UV: dose 1=60 sec, dose 2=120 sec.
For NaOCl: after 45-minute incubation with 16 (dose 1) or 32 (dose 2) mg/l NaOCl.
FIG. 3:
Antioxidant capacity of LDL in equivalent concentration of Trolox(copyright) (calibration substance) during Cu2+-induced oxidation.
FIG. 4:
Results of antioxidant capacity of plasma protein (ACP) measurements in healthy donors and cancer patients, 1 ASA=10 pmol/Asc/mg protein.
Mean values: 13.5 and 21.6 ascorbic acid equivalents (ASA); error probability p less than 0.0005.
Free radicals are associated with oxidative stress and can be generated by a variety of methods, i.e., physical (radiolysis, photolysis, electrolysis, etc.), physico-chemical (thermal decomposition of nitrogen compounds, photosensitized generation), chemical (Fe++/H2O2 system, KO2 decomposition, autoxidation of several compounds), and biochemical from individual enzymes (e.g., xanthine oxidase) to subcellular fractions (NADPH-consuming microsomes). The effect of the antioxidants can be detected by measurement of O2 consumption, light absorption, electrical conductivity, fluorescence, and chemiluminescence.
While investigating the mechanism of the therapeutic efficacy of ultraviolet irradiation of blood, it was surprisingly found that the antioxidant capacity of blood plasma increases during UV-B irradiation, rather than, as expected, declines. An in-depth investigation of this phenomenon has revealed that this increase in antioxidant capacity can be attributed to seven amino acids (cysteine, histidine, methionine, phenylalanine, serine, tryptophan and tyrosine). Their antioxidant capacity is unfolded during irradiation and increases in accordance with the dose. The antioxidant capacity of human serum albumin (HSA) undergoes similar changes during oxidation. As shown in FIG. 1, the antioxidant capacity (ACW) of human serum albumin increases during the course of irradiation with UV-light (xcex=254 nm). An increase of antioxidant capacity has also been found during irradiation of histidine solutions in which oxidative stress was induced by NaOCl or UV-light (xcex=254 nm) (FIG. 2). The effects of Cu2+-induced oxidation of LDL (low density lipoprotein) on its antioxidant capacity are shown in FIG. 3.
These so far unknown properties of blood plasma provide the basis for the determination of oxidative stress in living organisms by investigation of their protein-containing components.
The method according to the invention comprises the following steps:
A test sample is obtained from an organism, for example blood plasma or any other protein-containing substance. For example, blood is withdrawn from the cubital vein of a human. The blood plasma can be separated from the cells by, for example, centrifugation. The test sample, such as blood plasma, is then passed through a gel-chromatographic column. Such column can be a desalting column, for example, an Econo-Pac(trademark) 10DG column from Bio-Rad which contains Bio-Gel P-6 gel. The desalted test sample is eluted from the column with a phosphate buffer saline (PBS). The phosphate buffer saline can be prepared, for example, by adding 8.18 g NaCl and 3.58 g Na2HPO4x12H2O to 1 L H2O, and by adjusting the pH with HCl to 7.4.
The assay kit according to the present invention for the determination of oxidative stress in an organism by photochemiluminescence (PCL) investigations comprises a buffer and a photosensitizer. Preferably, the buffer has a basic pH value. More preferably the pH is between 7-14. Most preferably the pH is 10.6. The pH value includes all values therebetween, and especially including 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11, 11.5, 12, 12.5, 13, and 13.5. Alkali metal carbonates and earth alkali metal carbonates are preferably used as carbonates. More preferably, sodium carbonate is used as carbonate. The buffer concentrate can have a molarity of 0.1-0.3. Preferably, a 0.2 molar buffer solution in H2O is used. Different types of photosensitizer can be used, for example, riboflavine and methylene blue. Preferably luminol is used as a photosensitizer.
The eluate from the desalting column is added to the solution comprising the buffer and the photosensitizer. The solution can also contain deionized water.
The antioxidant capacity is preferably measured in a photochemiluminescence (PCL) measuring system. Free radicals can be generated in a free-radical generating system such as a photochemiluminescence (PCL) measuring system. Such system can be a Photochem(copyright) unit consisting of a cell for irradiation, a low pressure mercury lamp, a peristaltic mini-pump, a chemiluminescence measuring cell, and a personal computer. (Popov, I., Lewin, G. Antioxidative homeostasis: characterization by means of chemiluminescent technique. In: METHODS IN ENZYMOLOGY, Vol. 300, Eds. Packer, L. and Glazer, A. N., Academic Press, New York, 1999, p.p. 437-456).
Surprisingly, it was revealed that the antiradical (antioxidant) capacity of the proteins obtained from blood plasma increases in a dose-related manner after oxidatiye stress caused by both chemical (e.g. hypochlorite) and physical (e.g. ultraviolet light) factors. This capacity remains unchanged for at least 24 hours after treatment. Accordingly, it is possible to quantitatively determine the degree of oxidative stress in the organism after withdrawing blood and separation of proteins.
The oxidative capacity of a substance can be evaluated according to the degree of change of a parameter of the registered curve. This is the so-called lag-phase. the longer the lag-phase, the higher the antioxidative capacity of the substance. The antioxidative capacity of different substances is compared to a standard substance (calibration substant). Preferably ascorbic acid is used as standard substance. Accordingly, the antioxidative capacity is expressed in concentration units (mmol/l) of ascorbic acid, that has the same activity (lag-phase) in the measuring system.
The calibration proceeds as follows:
Solutions of different concentrations of ascorbic acid are prepared, containing, for example, 1, 2, 3, 4, and 5 nmol of ascorbic acid. A diagram for the dependence of the lag-phase from the concentration of the acid is then prepared based on the measurement of the antioxidative capacity of ascorbic acid. The lag-phase of measured samples can then easily be correlated to a specific concentration of the ascorbic acid. Accordingly, the measuring results can be evaluated in the PCL measuring system in equivalent concentrations of a suitable calibration substance (ascorbic acid or Trolox(copyright) consisting of a water soluble derivative of alpha-tocopherol), but also in absolute terms in seconds of the lag phase, of the point of inflection (maximum value of the first derivative) or as a percentage of inhibition (with the integral as evaluation parameter) of the PCL curves.
Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.